The Dynamic Range of Transcription

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The Dynamic Range of Transcription
Molecular Cell
The Dynamic Range of Transcription
Xavier Darzacq1 and Robert H. Singer2,*
Normale Supérieure, CNRS UMR 8541, 46 rue d’Ulm 75230, Paris Cedex 05, France
of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine,
1300 Morris Park Avenue, Bronx, NY 10461, USA
*Correspondence: [email protected]
DOI 10.1016/j.molcel.2008.05.009
In a recent issue of Molecular Cell, Gorski et al. (2008) demonstrate directly that polymerase assembly kinetics regulate Pol I transcription.
Within the last 5 years, tools have become
available to measure dynamic rates for
many biological processes in living cells.
One of the processes most amenable
to this approach is transcription since it
takes place at a defined location that can
be interrogated by fluorescence recovery
after photobleaching (FRAP) (Darzacq
et al., 2005, 2007 Dundr et al., 2002; Yao
et al., 2007). In this experimental design,
the transcription site, containing many
fluorescently labeled RNA polymerases
labeled by a fused fluorescent protein, is
bleached. Replacement by unbleached
polymerases is a direct measure of the
combination of events, including assembly at the promoter, initiation, and elongation (Darzacq et al., 2007; Dundr et al.,
2002). By fitting curves to these measurements, it is possible to dissect these various kinetic processes and model their
rate constants. Thus, the mechanisms of
transcriptional regulation can be more
fully understood.
Applied to both Pol I and Pol II, these
approaches have shown that transcription is inefficient (Darzacq et al., 2007;
Dundr et al., 2002; Yao et al., 2007).
Only a minor fraction of the polymerases
assembling at the promoter ultimately
make a transcript. This inefficiency could
have its foundation in the assembly of
a polymerase at the gene. Indeed, in
such a ‘‘neoassembly’’ model, polymerase subunits transiently interact and constantly assemble and disassemble on the
promoter (Figure 1, top). This scenario has
two possible outcomes: factor dissociation or complete polymerase assembly,
leading to promoter escape. Different
components of the Pol I enzyme were
found to interact with rDNA with different
residence times, strongly supporting the
neoassembly model (Dundr et al., 2002).
While this hypothesis challenges our
views of a more stable assembly, it can
explain the accessibility of the transcriptional machinery to compact chromatin
regions (Chen et al., 2005). Moreover,
findings in Drosophila showed that during
the heat shock transcriptional response,
the efficiency of polymerase recruitment
can evolve from an inefficient mode toward an efficient mode where enzymes
may be recycled internally among thousands of amplified genes (Yao et al.,
Knowing whether polymerases engage
DNA as preassembled, stable complexes
or if polymerase assembly occurs on DNA
is critical because how polymerases
assemble and engage DNA has implications for how transcription is regulated.
In the ‘‘preassembly’’ model, transcription
may be regulated at the level of initiation
and elongation of preassembled polymerases. Alternatively, in the ‘‘neoassembly’’
model, sequential steps in assembly may
be regulated, allowing transcription to
occur rapidly with a large dynamic range.
This is because in this scenario, an increase in the efficiency of any assembly
step could amplify transcriptional output
In a recent issue of Molecular Cell, Gorski et al. (2008) have demonstrated directly that polymerase assembly kinetics
regulate Pol I transcription. Transcription
of the ribosomal RNAs by Pol I is greatly
increased in S phase, and Gorski et al.
have tested the kinetics leading to this
increase using not only FRAP, but a
biochemical approach, the ChIP assay.
The results clearly show that the residence times of the components of the
Figure 1. Two Possible Modes of Polymerase Binding to DNA: Preassembly versus
Two models for polymerase transcriptional recruitment to genes. In the upper half, the polymerase subunits are recruited to the promoter where they assemble there into a functional enzyme. In the lower
half, the polymerases are preassembled and recruited as whole units helped by the interaction with
transcription factors. While these two models seem very similar, the upper one offers more opportunity
for regulation since the stoichiometry of the subunits can influence the initiation process.
Molecular Cell 30, June 6, 2008 ª2008 Elsevier Inc. 545
Molecular Cell
transcription complex are increased upon
transcriptional upregulation, thereby affecting the rate of entry into elongation.
The factors, TIF-1A and PAF53, which
mediate the interaction between the
preinitiation complex and the core polymerase, showed slower recovery in S
phase cells. Although these measurements translate into only about 2-fold increases in residence time, the modeling
demonstrates that this increase can
have profound effects on the assembly
rate and significantly improve the probability of entering into processive elongation and RNA production. Conversely,
mutants of these transcription factors
that are unable to promote initiation slow
down this process, specifically affecting
the kinetics of the particular subunit with
which they interact. This demonstrates
that the mode of action of these transcription factors is mediated by generating a local DNA-bound concentration of subunits
pushing the reaction toward assembly by
mass action. Regulation of these factors
during S phase appears to be due to the
usual suspects, namely various kinases
in the cell-cycle pathway.
All of this strongly supports a model
of sequential regulation of each of the
kinetic steps of transcription machinery
assembly rather than a model that would
provide a greater supply of assembled
complexes available to access the gene.
This likely occurs in Pol II transcription
as well, although unlike Pol I, these polymerases can pause during elongation of
the whole transcription unit, perhaps reflecting a continuous exchange of elongation factors not present in Pol I. While
initiation and transcriptional proximal regulation events are well understood biochemically and the sequential events of
promoter binding, preinitiation complex
formation, initiation, promoter escape,
and proximal pausing are well characterized (Core and Lis, 2008), none of
the kinetic rates observed in vivo can
unambiguously be attributed to one or
the other of these molecularly defined
events. More specific assays will enable
us identifying these steps in live cells.
The biggest challenge will be to unify molecular interpretations with kinetic live-cell
Improvements in the sampling rate with
higher sensitivity cameras, better optics,
and fluors will allow ever more temporal
detail to be delineated. This will provide
more exact rate constants and perhaps
546 Molecular Cell 30, June 6, 2008 ª2008 Elsevier Inc.
reveal even more components in the transcriptional cascade.
We thank John T. Lis, Olivier Bensaude, Thomas
Misteli, as well as members of the Darzacq and
Singer labs for their comments on the subject.
Supported by ANR-JCJC06_136138 to X.D. and
NIH grants to R.H.S.
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